Poster abstracts
Poster number 159 submitted by Xinyu Zhao
Abrogation of intronic polyadenylation: CRISPR/Cas9-based gene-editing to circumvent acquired resistance to DNA Topoisomerase II-targeted anticancer agents
Xinyu Zhao (Division of Pharmaceutics & Pharmacology, College of Pharmacy, The Ohio State University), Xinyi Wang (Division of Pharmaceutics & Pharmacology, College of Pharmacy, The Ohio State University), Sarah E. Eaton (Division of Pharmaceutics & Pharmacology, College of Pharmacy, The Ohio State University), Terry S. Elton (Division of Pharmaceutics & Pharmacology, College of Pharmacy, The Ohio State University), Jack C. Yalowich (Division of Pharmaceutics & Pharmacology, College of Pharmacy, The Ohio State University)
Abstract:
DNA topoisomerase IIα (TOP2α; 170 kDa, TOP2α/170) is a nuclear enzyme that plays a key role in chromosomal segregation at mitosis by catalyzing transient DNA double-stranded breaks, allowing replicated DNA duplexes to pass through one another. The clinical efficacy of TOP2α-targeted anticancer agents is often limited due to acquired drug resistance which is predominantly associated with decreased TOP2α/170 levels, attenuated drug-induced stabilization of TOP2α-DNA complexes, and reduced DNA damage/cytotoxicity.
We previously demonstrated decreased expression of TOP2α/170 protein in an acquired etoposide-resistant human leukemia K562 cell line (K/VP.5) due to the production of a novel C-terminal truncated 90 kDa isoform (TOP2α/90), the result of intronic polyadenylation (IPA) within Intron 19 (I19) of TOP2α mRNA. Circumvention of resistance was accomplished by gene-editing of the E19/I19 5'-splice site.
Truncated TOP2α mRNA transcript and protein have also been reported within a mitoxantrone-resistant subline (HL60/MX2) derived from human HL-60 leukemia cells as a result of I33 IPA, yielding a C-terminal truncated TOP2α/160 and reduced TOP2α/170 mRNA/protein. TOP2α/160 is predominantly distributed in the cytoplasm due to loss of a nuclear localization signal (NLS).
Utilizing 3'-Rapid Amplification of cDNA Ends (3'-RACE) and Sanger sequencing, IPA at I33 in HL60/MX2 cells was confirmed. In addition, use of THZ531, a CDK12 inhibitor, demonstrated a 30-fold increase in TOP2α/160 mRNA in HL60/MX2 cells compared to the parental HL60 cell line, further validating IPA in this resistant cell line.
CRISPR/Cas9 with homology-directed repair was used to successfully gene edit/mutate/enhance the intrinsically weak exon 33/intron 33 (E33/I33) splice-site in HL60/MX2 cells. Studies are underway to demonstrate abrogation of IPA, restoration of wild-type TOP2α/170 mRNA/protein level, and circumvention of anticancer drug sensitivity in these gene-edited cells.
Keywords: Intronic polyadenylation, DNA topoisomerase II